This research focuses on the role of neuronal migration in the development of neuronal diversity. Specifically, it tests the hypothesis that different substrates serve as guides for somatic and autonomic motor neuronal migration. Despite their common neurotransmitter, these two subclasses of cholinergic neurons exhibit different characteristics, and a goal of this investigation is to determine whether such phenotypic diversity is associated with differences in their migratory pathways. This hypothesis will be examined by pursuing the following specific aims: 1) Identification of the distribution patterns of somatic and autonomic motor neurons within the single, primitive motor column. Are the two subclasses initially intermixed in the primitive column, or is there an early spatial segregation at this stage of development? 2) Determination of the pathway for the secondary, dorsal translocation of autonomic motor cells. Is the dorsal translocation of these neurons from the primitive motor column guided by the axons derived from association interneurons? 3) Characterization of a tertiary movement of medially-located subsets of autonomic motor cells. Do autonomic cells that come to reside in medial locations migrate along the transverse dendritic bundles formed by lateral autonomic motor neurons? 4) Determination of whether various molecular markers of motor neurons are expressed prior to arrival at their final spatial addresses. Are subclass selective markers expressed by cells in the primitive motor column, or only after the establishment of separate autonomic and somatic motor columns? 5) Testing of possible causal relationships for the correlations observed in Specific Aims 1-4. Does a lesion-induced loss or an antibody perturbation of association fibers prevent the separation of the primitive motor column into somatic and autonomic columns? Experiments seeking answers to these questions will utilize retrograde labeling techniques, double label immunocytochemical analyses, combined retrograde tracer and immunocytochemical studies, and a newly developed, organotypic in vitro preparation of embryonic rat spinal cord. One of the hallmarks that distinguishes the nervous system is the degree of phenotypic diversity exhibited by its cellular constituents. Understanding how this diversity is generated is of key importance to both normal brain function and a variety of neurological disorders.